Biodegradation of ochratoxin A by fungi isolated from grapes

Ochratoxin A is a mycotoxin present in several food products for which levels should be reduced. Chemical, physical, and biological methods have been proposed for the detoxification of mycotoxins, biological methods being the more promising ones. In this report, filamentous fungi isolated from Portuguese grapes were assessed for ochratoxin A degradation capabilities. It was observed that 51 of the 76 tested strains, predominantly aspergillus species, were able to degrade more than 80% of ochratoxin A added to the culture medium and that the most potent species (more than 95% of initial amount) were the black aspergilli, A. clavatus, A. ochraceus, A. versicolor, and A. wentii. Other fungi frequently isolated from grapes, such as Alternaria, Botrytis, Cladosporium, and Penicillium, also showed significant degradation capabilities. It was observed that the compounds obtained from the degradation of ochratoxin A by black aspergilli and by A. ochraceus and A. wentii strains were different.     

Volatile compounds of Aspergillus strains with different abilities to produce ochratoxin A

Volatile compounds emitted by Aspergillus strains having different abilities to produce ochratoxin A were investigated. Thirteen strains of Aspergillus ochraceus, three belonging to the A. ochraceus group, and eight other species of Aspergillus were examined for their abilities to produce volatile compounds and ochratoxin A on a wheat grain medium. The profiles of volatile compounds, analyzed using SPME, in all A. ochraceus strains, regardless of their toxeginicity, were similar and comprised mainly of 1-octen-3-ol, 3-octanone, 3-octanol, 3-methyl-1-butanol, 1-octene, and limonene. The prevailing compound was always 1-octen-3-ol. Mellein, which forms part of the ochratoxin A molecule, was found in both toxigenic and nontoxigenic strains. Volatile compounds produced by other Aspergillus strains were similar to those of A. ochraceus. Incubation temperatures (20, 24, and 27 degrees C) and water content in the medium (20, 30, and 40%) influenced both volatile compounds formation and ochratoxin A biosynthesis efficiency, although conditions providing the maximum amount of volatiles were different from those providing the maximum amount of ochratoxin A. The pattern of volatiles produced by toxigenic A. ochraceus strains does not facilitate their differentiation from nontoxigenic strains.     

Fate of ochratoxin A during vinification of Semillon and Shiraz grapes

Semillon and Shiraz grapes containing ochratoxin A (OA) were obtained by inoculation of bunches on the vine with Aspergillus carbonarius. Citric acid content was greater in the inoculated grapes than in healthy grapes. Samples were collected throughout vinification of these grapes and the OA content was quantified using a stable isotope dilution liquid chromatographic-tandem mass spectrometric method. The mass of processed and waste streams during vinification was also noted. Reduction in the amount of OA in juice and wine occurred at every solid-liquid separation stage. The OA concentration (microg/kg) in white and red wine after racking was 4% and 9%, respectively, of that in crushed grapes. This corresponds to 1% and 6% of the total OA content that was initially present in the inoculated grapes. The OA content was divided between solid and liquid phases at each stage of vinification. OA did not appear to be transformed either chemically or biologically by yeast during fermentation, rather was discarded with the marc, juice lees, and gross lees.     

Effect of lime-induced leaf chlorosis on ochratoxin A, trans-resveratrol, and epsilon-viniferin production in grapevine (Vitis vinifera L.) berries infected by Aspergillus carbonarius

Berries of Vitis vinifera L. cv. Merlot, grown on a neutral or calcareous soil, were infected, at phenological phases of veraison and ripening, by a conidial suspension of Aspergillus carbonarius to control ochratoxin A production and trans-resveratrol- and epsilon-viniferin-induced synthesis as affected by the soil lime content. Chlorosis occurrence was evaluated by a visual rating scale at veraison, and the leaves from vines growing on the calcareous soil showed the typical yellowing, whereas those grown on the neutral soil were dark green. Berry mineral element yield was recorded at veraison and ripening. Infection symptoms on berries were more severe at ripening in bunches collected from vines grown in calcareous soil. Ochratoxin A concentration increased at phenological phase of veraison in berries harvested from vines cultivated in calcareous soil. A. carbonarius enhanced trans-resveratrol and epsilon-viniferin production in infected berries more than in the control samples. Moreover, at veraison their concentration in the berries collected from vines grown in calcareous soil was greater than that recorded from berries collected from vines grown in the neutral soil. The lowest symptom severity was observed on berries containing the highest copper concentration.     

Fungal flora and ochratoxin a production in grapes and musts from france

Eleven samples of grapes and musts used in red table wines were investigated for the occurrence of potential ochratoxin A (OTA)-producing molds. From these samples, 59 filamentous fungi and 2 yeasts were isolated. Among the 30 genera isolated, Deuteromycetes were the most frequent (70%) followed by Ascomycetes (10%). Six of the eleven grapes samples were contaminated by potentially ochratoxinogenic strains (Penicillium chrysogenum and Aspergillus carbonarius). When cultivated in vitro on solid complex media, the 14 strains of A. carbonarius produced OTA. No other species produced OTA under the same conditions. Among must samples, eight of eleven were found to be contaminated by OTA (concentrations from <10 to 461 ng/L). There is a strong correlation between the presence of ochratoxin-producing strains on grapes and OTA in musts. These findings should be connected with the OTA contamination of human blood in these areas and in France.     

Ultraviolet-C and induced stilbenes control ochratoxigenic Aspergillus in grapes

This study investigated the efficacy of ultraviolet-C (254 nm) and induced stilbenes to inhibit Aspergillus carbonarius and Aspergillus tubingensis and control ochratoxin A production in grapes. In addition, the stilbene synthesis as a response to UV-C treatment and to infection of ochratoxigenic Aspergillus was compared. The initial microbial inactivation by a previously optimized UV-C illumination protocol for increasing trans-resveratrol content in grapes (50 W/m (2), 40 cm, 60 s) was similar on undamaged and damaged grapes, achieving 1.2 and 1.3 log conidia/100 g reductions, respectively. After 5 days of storage at 22 degrees C, UV-C treatment and the stilbenes induced by UV-C inhibited ochratoxigenic Aspergillus growth in undamaged grapes. UV-C elicited the biosynthesis of trans-resveratrol, while microbial infection and tissue damage triggered the biosynthesis of trans-piceid. trans-Resveratrol was not synthesized as a consequence of ochratoxigenic Aspergillus contamination. However, when trans-resveratrol was synthesized by UV-C, it contributed to inhibiting the development of ochratoxin A producing aspergilli. Furthermore, UV-C treatment also contributed to decrease ochratoxin A production by ochratoxigenic aspergilli. Therefore, UV-C is a promising emerging technology either for reducing the potential ochratoxigenic risk in grapes, which is of particular interest to the wine industry, and also for increasing trans-resveratrol content of grapes, which would provide an added value to the wine.     

Drying of Pedro Ximenez grapes in chamber at controlled temperature and with dipping pretreatments. Changes in the color fraction

The drying of Pedro Ximenez grapes in chamber at a controlled temperature of 40 or 50 degrees C is studied. Compared to traditional sun-drying, the chamber-drying shortened the drying time by about 40% at 50 degrees C. In color terms, the musts obtained from grapes dried at 50 degrees C were closer in CIELab coordinates to those obtained by sun-dried grapes, with similar h(ab) values and slightly lower L* and C(ab)*. To shorten further the drying times at 50 degrees C, the grapes were dipped in olive oil or ethyl oleate emulsions containing potassium carbonate. The ethyl oleate pretreatment shortened additionally the drying time by about 25%, providing musts with chroma, lightness, and hue similar to those without grape pretreatment. In general, except for the phenolic compounds corresponding to the drying with ethyl oleate pretreatment, most of these compounds in the remainding conditions studied increased to a lesser extent than expected because of water losses of the grapes during drying, revealing degradation reactions.     

Occurrence of naphtho-gamma-pyrones- and ochratoxin A-producing fungi in French grapes and characterization of new naphtho-gamma-pyrone polyketide (aurasperone G) isolated from Aspergillus niger C-433

A survey on the occurrence on grape of fungi species in 2001 and their capability to produce ochratoxin A (OTA) and naphtho-gamma-pyrones (NGPs) was conducted in different vineyards from several French viticulture regions. The total numbers of fungal isolates, from setting to harvest, were 732. The Aspergillus genus was essentially represented by section Nigri (98.53%) and it was predominant (74.72%) when compared to Penicillium (25.27%). Approximately one third (30.46%) of the fungal isolates were OTA producers, and 94.17% belong to black aspergilli; Aspergillus carbonarius was the main OTA producer. Moreover, 8.33% of isolates (belong to A. carbonarius and A. niger) were NGP producers. However, none of the Penicillium spp. or other Aspergillus spp. isolates can produces NGP derivatives under the conditions used. No other study on NGPs production by fungi isolated from grapes has been reported. In the second part, a novel NGP, named aurasperone G (1), was isolated from the fermentation broth of the culture extracts of Aspergillus niger C-433, strain producer of OTA, along with the known compound aurasperone F (2). The chemical structure of the new polyketide was proposed based on complete (1)H and partial (13)C, COSY, HMQC, 1D NOE NMR spectra as well as UV and MS spectra. This new NGP was not reported before in nature or prepared synthetically.     

Fate of isoxaflutole in soil under controlled conditions

Isoxaflutole (IFT, 5-cyclopropyl-1,2-oxazol-4yl-alpha,alpha,alpha-trifluoro-2-mesyl-p-tolyl ketone) is a new pre-emergence proherbicide used in maize and sugarcane. Its two main derivatives are a diketonitrile derivative, 2-cyano-3-cyclopropyl-1-(2-methanesulfonyl-4-trifluoromethylphenyl)propane-1,3-dione, called DKN, and a benzoic acid derivative, 2-methanesulfonyl-4-trifluoromethylbenzoic acid, called BA. Few data are available of the factors influencing the degradation of IFT in soil, and the purpose of the present work was to determine the relative importance of, and factors affecting, the degradation of IFT in soil. Experiments were conducted on five soils with distinct physicochemical characteristics, at different temperatures and moisture contents in biotic and abiotic conditions. The isomerization of IFT to DKN is rapid, increasing with higher moisture contents and higher temperatures. It depends strongly on pH and is governed by chemical processes. The degradation of DKN to BA appeared to be essentially due to the biological activity of the soil.     

Occurrence of ochratoxin A- and aflatoxin B1-producing fungi in Lebanese grapes and ochratoxin a content in musts and finished wines during 2004

This paper reports the results of an extensive survey on the occurrence of filamentous fungi isolated from wine-grapes in Lebanon and to test their ability to produce ochratoxin A (OTA) and aflatoxin B1 (AFB1) on CYA culture medium, in order to assess their potential for producing these mycotoxins on grapes. From the 470 grapes samples taken during season 2004, 550 fungi strains were isolated with 490 belonging to Aspergillus spp. and 60 belonging to Penicillium spp. All these isolated fungi starins were tested for their ability to produce OTA and AFB1. Aspergillus carbonarius shows that it is the only species able to produce OTA with a production percentage reaching 100% and a maximum concentration of 52.8 microg/g of Czapek yeast extract agar (CYA). In its turn, Aspergillus flavus was considered as the only AFB1-producing species with production percentage of 45.3% and a maximum concentration reaching 40 microg/g CYA. A total of 47 handmade musts produced from the collected grapes were also analyzed in order to correlate the presence of OTA in must and the occurrence of filamentous fungi on grapes; 57.4% were contaminated with OTA at low level with concentrations ranging between 0.011 and 0.221 microg OTA L(-1). The analysis of these must samples was not performed with regard to AFB1. Seventy samples of finish red wine were also assayed for OTA content. The results showed that 42 of the tested samples (60%) were found to be positive for OTA with low levels (0.012-0.126 microg OTA L(-1)).     

Changes in hydrophilic and lipophilic antioxidant activity in relation to their phenolic composition during the chamber drying of red grapes at a controlled temperature

The purpose of this work was to study the variation of phenol compounds, as measured by HPLC, during the chamber drying under controlled temperature conditions of red grapes of the Merlot and Tempranillo varieties in relation to antioxidant activity. Both lipophilic and hydrophilic antioxidant activities in these grapes increased during the drying process; the former was measured via proton transfer in the coupled oxidation reaction between linoleic acid and β-carotene, and the second via electron transfer in the DPPH assay. The hydrophilic component was invariably greater in Tempranillo grapes, and so was the lipophilic component in Merlot grapes. Only the increase in hydrophilic antioxidant activity obtained a significant correlation with the phenolic compounds during the drying process. However, based on the phenolic fraction analysis, this result was primarily due to phenolic polymers and, to a lesser extent, also to phenolic acids, flavans, and some flavonols and anthocyans.     

Isolation,selection and evaluation of Rhizobium under controlled conditions

Abstract

Rhizobia were screened in growth chamber and greenhouse tests with the host plants: bean (Phaseolus vulgaris L.), lentil (Lens esculenta Moench.), cowpea (Vigna unguiculata (L.) Walp) and peanut (Arachis hypogaea L.). Rhizobial isolates varied in effectiveness, time to nodulation, and host plant specifities. Initial screening procedures in plastic growth pouches allowed selection of infective strains of Rhizobium. These tests enabled the selection of a small number of highly effective isolates for more critical evaluation. Highly significant correlations (p=0.01) were obtained between shoot dry weight and total nitrogen, suggesting that the technique was applying sufficient selection pressure to differentiate variation in N₂ fixation among strains of Rhizobium. Results indicate that highly effective rhizobia can be efficiently selected under controlled conditions following a step‐wise procedure.     

Nitrous oxide emission from animal manures applied to soil under controlled conditions

Animal manures may differ strongly in composition and as a result may differ in the emission of N₂O following application to soil. An incubation study was carried out to assess the effects of type of mineral N fertilizer and manure, application technique and application rate on N₂O emission from a sandy soil with low organic matter content. Fluxes of N₂O were measured 30 times over a 98-day period. The total N₂O emission from mineral N fertilizer ranged from 2.1 to 4.0% of the N applied. High emissions were associated with manures with high contents of inorganic N, easily mineralizable N and easily mineralizable C, such as liquid pig manure (7.3-13.9% of the N applied). The emission from cattle slurries ranged from 1.8 to 3.0% and that of poultry manures from 0.5 to 1.9%. The total N₂O emission during the experimental period tended to increase linearly with increasing N application rate of NH₄NO₃ and liquid pig manure. The N₂O emission from surface-applied NH₄NO₃ was significantly smaller than that following the incorporation of NH₄NO₃ in the soil. The N₂O emission from pig manure placed in a row at 5 cm depth was significantly higher than from surface-application and other techniques in which manure was incorporated in the soil. The results show that modification of the composition and application technique may be tools to mitigate emission of N₂O.     

Strong functional stability of soil microbial communities under semiarid Mediterranean conditions and subjected to long-term shifts in baseline precipitation

We investigated the effect of soil microclimate on the structure and functioning of soil microbial communities in a Mediterranean Holm-oak forest subjected to 10 years of partial rain exclusion manipulations, simulating average drought conditions expected in Mediterranean areas for the following decades. We applied a high throughput DNA pyrosequencing technique coupled to parallel measurements of microbial respiration (R_H) and temperature sensitivity of microbial respiration (Q₁₀). Some consistent changes in the structure of bacterial communities suggest a slow process of community shifts parallel to the trend towards oligotrophy in response to long-term droughts. However, the structure of bacterial communities was mainly determined by short-term environmental fluctuations associated with sampling date (winter, spring and summer) rather than long-term (10 years) shifts in baseline precipitation. Moreover, long-term drought did not exert any chronic effect on the functioning of soil microbial communities (R_H and Q₁₀), emphasizing the functional stability of these communities to this long-term but mild shifts in water availability. We hypothesize that the particular conditions of the Mediterranean climate with strong seasonal shifts in both temperature and soil water availability but also characterized by very extreme environmental conditions during summer, was acting as a strong force in community assembling, selecting phenotypes adapted to the semiarid conditions characterizing Mediterranean ecosystems. Relations of climate with the phylogenetic structure and overall diversity of the communities as well as the distribution of the individual responses of different lineages (genera) to climate confirmed our hypotheses, evidencing communities dominated by thermotolerant and drought-tolerant phenotypes.     

The response of drought-tolerant sugar beet to salinity stress under field and controlled environmental conditions

Abstract

Thirty-four sugar beet drought-tolerant half-sib families were screened in greenhouse experiment under saline (16?dSm^?1) and normal conditions, and a subset of 10 tolerant genotypes were selected for evaluating of yield-related traits, Na⁺, K⁺, and α-amino-N nitrogen contents under saline field condition (12?dSm^?1). Drought-tolerant genotypes showed a different tolerance level under salt stress. Significant correlation was observed between EP2_s and STIEP2 in greenhouse with root yield (RY) in saline field condition. It should be possible that a simple measurement at 16?dSm^?1 in the greenhouse can be used as main part of sugar beet salt-tolerant breeding program. In total, genotypes SBSI-DR I-HSF14-P.23 (# 16) and 436B-HSF9-P.27 (# 26) were recognized as salt- and drought-tolerant genotypes with low Na⁺ and K⁺ contents in root and high white sugar content (WSC) and RY in field. A larger panel of genotypes to validate this kind of association is necessary.     

Substrate utilization by Aspergillus flavus in inoculated whole corn kernels and isolated tissues

Utilization of the major corn (Zea mays) reserve materials (free saccharides, starch, triglycerides, and zein) was monitored during infection of detached kernels by Aspergillus flavus (A. flavus) over a 12-day period. Inoculated whole kernels were compared to noninoculated kernels. Concentrations of sucrose and raffinose in inoculated seed decreased to nearly zero at 6 days, whereas concentrations of these saccharides in noninoculated seed dropped at a considerably slower rate, and significant levels remained at the end of the incubation period. Triglyceride concentrations remained unchanged in the noninoculated seed but dropped continuously after 2 days in the inoculated seed. Starch and zein concentrations did not change during the 12-day incubation period. Aflatoxin B1 was first detected after 2 days and increased to about 20 microg/g (20,000 ppb) after 12 days. Very low aflatoxin concentrations were detected in the noninoculated seed. Significant concentrations of erythritol, arabitol, and mannitol were produced during infection, with peak concentrations occurring at 8 days. Whole seed and germ tissue appeared to support good fungal growth and aflatoxin production, whereas ground tissues and endosperm did not. A. flavus preferentially utilized saccharides as initial carbon substrates followed by triglycerides. When invading nonwounded corn kernels, the fungus selectively targets the germ tissue where these materials are localized in the highest concentrations.     

Changes of redox and pH conditions in a flooded soil amended with glucose and manganese oxide or iron oxide under laboratory conditions

The changes of Eh and pH in soil suspension (Ah-horizon of a Mollic Gleysol) and Mn²⁺ or Fe²⁺ concentrations in the equilibrium soil solution at different levels of glucose (0%, 0.5% and 1%) and MnO₂ (0%, 0.025%, 0.05% and 0.1%) or Fe₂O₃ (0%, 0.025%, 0.05% and 0.1%) were examined. It was found that the degree of Mn- and Fe-reduction in soil depends mainly on the presence and the amount of an easily decomposable carbon source and to a minor degree on the content of native or added forms of MnO_O2 or Fe₂O₃ in the soil. Theoretical relationships between the water soluble manganese and iron and the Eh and pH values have been verified, when the observed initial drop of Eh was eliminated. It was found that the water soluble manganese content was described best by the Mn₂O₃/Mn²⁺ redox system, and that of iron by the Fe₃ (OH)₃/Fe²⁺ system.     

Effect of roasting conditions on reduction of ochratoxin a in coffee

A commercial lot of green coffee, naturally contaminated with ochratoxin A (OTA), was roasted under various conditions, and the effects on its final OTA content were determined. Precautions were taken in sampling the coffee to cope with OTA inhomogeneity. The roasting conditions were kept within the range of commercial practice. Roasting time was varied from 2.5 to 10 min, and the roast color varied from light medium to dark. The differences in OTA reduction between the different levels of roasting times and colors did not reach statistical significance. However, for all roasting conditions, the reduction was highly significant, 69% reduction over the combined results. In total, nine studies by various authors about OTA reduction during coffee roasting are now available. Seven out of these nine reported that the relevant range of OTA reductions was between 69 and 96%. Among these seven,are all four studies that reported using naturally contaminated beans, a sampling procedure adapted to mycotoxin inhomogeneity, and roasting conditions within the range of actual practice. Three different explanations are available for this reduction: physical removal of OTA with chaff, isomerization at the C-3 position into another diastereomer, and thermal degradation with possible involvement of moisture. All three explanations may play a partial role in the OTA reduction during coffee roasting.     

不同控制灌溉方式下稻田土壤盐分动态变化研究

对不同控制灌溉方式下稻田土壤盐分在水稻各生育期的动态变化规律进行了深入分析,并结合各生育期水稻冠层叶面积指数和稻田腾发量对土壤盐分动态成因进行了探讨。研究结果表明：控制灌溉或控制灌溉+淋洗条件下,各水稻生育期土壤含盐量均有不同程度降低,表现为随着淋洗水量的增加土壤含盐量逐渐降低的规律性,试验条件下不同处理间的差异不太显著;试验各灌溉方式并未引起土壤盐分显著积聚,并且具有重要的节水增产效益,其中水稻控制灌溉(即处理1)从节水增产角度而言是稻田较佳的灌溉管理方式。本项研究内容及其成果对于指导水稻灌溉管理实际具有重要的理论指导意义。